More than half of all proteins contain metal ions. A large percentage of those contain two metal ions (usually first-row transition metals or magnesium) connected by a bridging ligand (usually a carboxylate group). Most of these are enzymes, and they catalyze a great variety of different chemical reactions, ranging from hydrolysis to oxidation to isomerization to biopolymer synthesis. Yet little is understood about how the two metal ions work together in catalysis, or how the protein environment modulates the intrinsic chemical reactivity of the dimetal center. The objective of this proposal is to discover the general features common to all bridged bimetalloenzyme mechanisms as well as the specific effects of the rest of the protein on the chemistry of the metal cluster. We have selected two enzymes, Streptomyces olivochromogenes xylose isomerase (Xyl) and Aeromonas proteolytica aminopeptidase (AAP), as primary model systems for this investigation. Xylose isomerase uses a bridged dimagnesium center to catalyze sugar ring-opening followed by aldose-ketose interconversion, a reaction of great importance in the food industry. Aminopeptidase uses a dizinc center to hydrolyze the N-terminal amino acid from peptides and proteins;members of its family are targets for antimicrobial, antiviral and antitumor drugs. We intend to employ a range of techniques, including site-directed mutagenesis, kinetic analysis, inhibitor design and synthesis, ultra-high resolution X-ray crystallography, neutron diffraction, and combined quantum mechanics/molecular mechanics simulations to probe the role of the second shell residues in AAP and the role of the bridging ligand in Xyl. Crystals of both enzymes diffract X-rays to beyond 1 A resolution, allowing us to obtain extremely precise interatomic parameters and to determine the spatial distribution of both individual atomic vibrations and collective motions of groups of atoms. These data will be used as input into quantum mechanical and other calculations, allowing us to see how the rest of the protein affects the electronic distribution and chemical properties of the dimetal center. We already have evidence that mutation of at least one of the second shell ligands in AAP causes a significant change in the chemical properties of the bridged dizinc center: in the S228A mutant, the enzyme is much more sensitive than the wild-type protein to inhibition by sulfur containing compounds. In an additional specific aim, we will help develop a new method of refining protein crystal structures, one that incorporates a quantum mechanical potential. In addition, since we have just solved the structure of a bacterial quorum-sensing acyl-homoserine lactone hydrolase, we will carry out similar experiments and calculations on this dizinc enzyme, which has an unusual monodentate bridge.